Effects of Eosin Y on the Catalytic and Functional Activities of Mg²⁺,ATP-Dependent Calcium Pump of Smooth Muscle Cell Plasma Membrane

N. N. Slinchenko, N. F. Bratkova, S. A. Kosterin^*, V. P. Zimina, and I. G. Chernysh

Department of Muscle Biochemistry, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, ul. Leontovicha 9, Kiev, 252030 Ukraine; fax: (044) 229-6365; E-mail: Kinetic@biochem.Kiev.ua

^* To whom correspondence should be addressed.

Received October 16, 1997; Revision received March 3, 1998

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Experiments with highly purified preparations of Ca²⁺,Mg²⁺-ATPase solubilized from sarcolemma and a fraction of inside-out sarcolemmal vesicles were performed to study the kinetics of inhibitory effects of eosin Y (0-100 µM) on the catalytic and transport activity of Mg²⁺,ATP-dependent calcium pump of myometrial cell plasma membrane. For both the Ca²⁺,Mg²⁺-dependent ATP hydrolysis and the Mg²⁺,ATP-dependent accumulation of Ca²⁺ the apparent inhibitory constant K_i was 0.8 µM. However, eosin Y used at concentrations of up to 100 µM had absolutely no effect on Na⁺-Ca²⁺ exchange in the plasma membrane fraction. This inhibitor decreased the turnover rate of purified Ca²⁺,Mg²⁺-ATPase determined both by Mg²⁺ and ATP. However, the affinity of purified Ca²⁺,Mg²⁺-ATPase for Mg²⁺ increased, and its affinity for ATP decreased in the presence of eosin Y. In the case of Mg²⁺,ATP-dependent Ca²⁺ transport, eosin Y decreased the turnover rate of the calcium pump whose affinity for Mg²⁺ and ATP displayed virtually no dependence on the presence of the inhibitor in the incubation medium. The possibility of the use of eosin Y in model experiments for identification of Mg²⁺,ATP-dependent and Na⁺-dependent calcium fluxes from smooth muscle cells into the intercellular milieu is discussed.

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KEY WORDS: Ca²⁺,Mg²⁺-ATPase, calcium pump, eosin Y, plasma membrane, smooth muscle cells, myometrium

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